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Abstract:

A composition for treating or controlling an ocular disease or condition
comprises a dissociated glucocorticoid receptor agonist ("DIGRA"), which
disease or condition has an etiology, or results, in inflammation. The
composition can optionally include an anti-inflammatory agent, an
anti-infective agent, or both. The composition can be formulated for
topical application, injection, or implantation in an affected eye to
treat or control the ocular inflammatory disease or condition.

Claims:

1. A method for treating or controlling an ocular inflammatory disease,
condition, or disorder, comprising administering a composition comprising
a DIGRA, a prodrug thereof, or a pharmaceutically acceptable salt or
ester thereof to an affected eye of a subject in need of such treatment
or control, wherein the DIA has Formula I ##STR00004## wherein A and Q
are independently selected from the group consisting of unsubstituted and
substituted aryl and heteroaryl groups, unsubstituted and substituted
cycloalkyl and heterocycloalkyl groups, unsubstituted and substituted
cycloalkenyl and heterocycloalkenyl groups, unsubstituted and substituted
cycloalkynyl and heterocycloalkynyl groups, and unsubstituted and
substituted heterocyclic groups; R1 and R2 are independently
selected from the group consisting of hydrogen, unsubstituted
C1-C15 linear or branched alkyl groups, substituted
C1-C15 linear or branched alkyl groups, unsubstituted
C3-C15 cycloalkyl groups, and substituted C3-C15
cycloalkyl groups; R3 is selected from the group consisting of
hydrogen, unsubstituted C1-C15 linear or branched alkyl groups,
substituted C1-C15 linear or branched alkyl groups,
unsubstituted C3-C15 cycloalkyl and heterocycloalkyl groups,
substituted C3-C15 cycloalkyl and heterocycloalkyl groups, aryl
groups, heteroaryl groups, and heterocyclylic groups; B comprises a
carbonyl, amino, divalent hydrocarbon, or heterohydrocarbon group; E is
hydroxy or amino group; and D is absent or comprises a carbonyl group,
--NH--, or --NR'--, wherein R' comprises an unsubstituted or substituted
C1-C15 linear or branched alkyl group; and wherein R1 and
R2 together may form an unsubstituted or substituted
C3-C15 cycloalkyl group; wherein DIGRA, a prodrug thereof, or a
pharmaceutically acceptable salt or ester thereof is present in an amount
effective to treat or control said ocular inflammatory disease,
condition, or disorder; wherein the method provides a lower risk of
inducing increased IOP than a method using a glucorticosteroid, and
wherein said lower risk results from a lower production of myocilin from
trabecular meshwork.

3. The method of claim 2, wherein the DIGRA has Formula I ##STR00005##
wherein A and Q are independently selected from the group consisting of
aryl and heteroaryl groups substituted with at least a halogen atom,
cyano group, hydroxy group, or C1-C10 alkoxy group; R1,
R2, and R3 are independently selected from the group consisting
of unsubstituted and substituted C1-C5 alkyl groups; B is a
C1-C5 alkylene group; D is the --NH-- or --NR'-- group, wherein
R' is a C1-C5 alkyl group; and E is the hydroxy group.

4. The method of claim 2, wherein the DIGRA has Formula I ##STR00006##
wherein A comprises a dihydrobenzofuranyl group substituted with a
fluorine atom; Q comprises a quinolinyl or isoquinolinyl group
substituted with a methyl group; R1 and R2 are independently
selected from the group consisting of unsubstituted and substituted
C1-C5 alkyl groups; B is a C1-C3 alkylene group; D is
the --NH-- group; E is the hydroxy group; and R3 comprises a
trifluoromethyl group.

6. The method of claim 5, wherein the DIGRA has Formula IV ##STR00008##

7. The method of claim 6, wherein said composition further comprises an
anti-inflammatory agent is selected from the group consisting of NSAIDs,
PPAR agonists, combinations thereof, and mixtures thereof.

Description:

[0002]The present invention relates to compositions and methods for
treating or controlling ocular inflammation. In particular, the present
invention relates to compositions that comprise dissociated
glucocorticoid receptor agonists ("DIGRAs") and methods for the treatment
or control of ocular inflammation using such compositions, which
compositions and methods provide lower risk of increased intraocular
pressure.

[0004]Glucocorticoids ("GC," also herein referred to as corticosteroids)
are often prescribed to treat a variety of ocular conditions having an
inflammatory or neovascular component, such as macular edema, "wet"
age-related macular degeneration, uveitis, and complications of surgery.
The therapeutic benefit of GC is due to pleiotropic modulation and
mobilization of multiple intracellular signaling pathways, encompassing
predominantly transrepressive effects of the steroid-nuclear receptor
complex that interfere with elements governing transcription of selected
genes. One of the adverse events commonly associated with glucocorticoid
therapy, regardless of route of administration, is an elevation of
intraocular pressure ("IOP") that may lead to glaucoma, a side-effect
assumed to result from transactivation of a gene or genes unrelated to
the indication being treated. Some patients receiving ocular GC may
exhibit no effect, while others, classified as responders, demonstrate a
range of documented increases in IOP, attributed to several risk factors,
including age, history of primary open-angle glaucoma ("POAG"), and
genetic predisposition.

[0005]POAG is characterized by high TOP, resulting from impaired efflux of
aqueous fluid through the trabecular meshwork ("TM"). Since the
juxtacanalicular region ("JCT") of the TM abutting the inner wall
endothelium of Schlemm's canal is the likely site of resistance to
outflow under normal physiological conditions, structural and biochemical
changes in the JCT would be expected to affect IOP. A feature shared by
both POAG and steroid-induced glaucoma is the accumulation of
extracellular matrix ("ECM") and other material ("plaque") in the JCT,
consisting of abnormal aggregates of macromolecules obstructing the
outflow pathway and raising IOP. As with many other non-ocular cells and
tissues that have been examined, the TM is susceptible to GC-induced ECM
changes, demonstrated experimentally and in clinical samples.

[0006]Myocilin is a protein normally detected in eye tissues, and whose
constitutive expression is most pronounced in the TM, both intra- and
extracellularly. The discovery that mutations in the myocilin gene (MYOC)
give rise to selected forms of POAG and juvenile open-angle glaucoma
eventually directed attention to the roles of wild-type myocilin in eye
health and disease. An apparently unique--and diagnostic--property of TM
cells in vitro and in situ is the overexpression of myocilin in response
to GC. The precise functional role of myocilin is not understood, but
GC-enhanced TM expression of myocilin has raised the possibility that
this protein has an etiological role in steroid-induced glaucoma. Besides
effects on TM cell internal structure and function, as assessed in organ
cultured material, GC treatment may induce excessive myocilin synthesis
and secretion by these cells, culminating with deposition of the protein
in the ECM of the outflow pathway, and hence, elevating IOP.
Pharmacologic doses of dexamethasone ("DEX") elicit elevated expression
of myocilin in cultured TM cells from normal human donors, shown by
analysis of myocilin mRNA or through immunochemical detection of soluble
myocilin released into culture media. Irrespective of whether or not
these changes underlie a direct role for myocilin in the pathophysiology
of any form of glaucoma, drug-induced elevations of this protein could be
considered a surrogate indicator of risk for secondary glaucoma.

[0007]There is considerable interest in non-steroidal GC receptor (GR)
agonists that, by virtue of their structures and of the specific
conformational changes they generate upon binding to the GR, may exhibit
partial dissociation with respect to transactivation and transrepression
of selected genes normally affected by GCs. Molecules with these distinct
biochemical profiles may offer an improved clinical safety profile
compared to steroidal GR agonists routinely used in the clinic. Human TM
cells have been widely employed as an in vitro model to study responses
to GCs.

[0008]Therefore, there is a continued need to provide improved
pharmaceutical compounds, compositions, and methods to treat or control
ocular inflammatory diseases, conditions, or disorders that provide lower
risk of increased IOP than a composition and a method using a prior-art
glucocorticoid used to treat or control the same diseases, conditions, or
disorders.

SUMMARY OF THE INVENTION

[0009]As used herein, the term "control" also includes reduction,
alleviation, amelioration, and prevention.

[0010]In general, the present invention provides compounds, compositions,
or methods for treating or controlling an ocular inflammatory disease,
disease, condition, or disorder. Such an inflammatory disease, condition,
or disorder has etiology in, or produce, inflammation.

[0011]In one aspect, the compounds, compositions, methods of the present
invention provides a lower risk of increased IOP than a composition and
method using a prior-art GC to treat or control the same diseases,
conditions, or disorders.

[0013]In still another aspect, such inflammatory diseases, conditions, or
disorders result from an infection caused by bacteria, viruses, fungi, or
protozoans.

[0014]In yet another aspect, the compositions comprise at least a mimetic
of a glucocorticoid for treating or controlling such diseases,
conditions, or disorders.

[0015]In yet another aspect, a pharmaceutical composition for treating or
controlling an inflammatory disease, condition, or disorder comprises at
least a dissociated glucocorticoid receptor agonist ("DIGRA"), a prodrug,
or a pharmaceutically acceptable salt or ester thereof.

[0017]In still another aspect, such a formulation, system, or device is
applied or provided to the anterior segment of the eye.

[0018]In still another aspect, such a formulation, system, or device is
applied or provided to the posterior segment of the eye.

[0019]In a further aspect, said increased IOP is demonstrated in vitro or
in vivo.

[0020]In yet another aspect, said increased IOP results from an increased
resistance of fluid outflow through the trabecular meshwork.

[0021]In still another aspect, such increased resistance results from
increased expression and accumulation of myocilin in the trabecular
meshwork.

[0022]In another aspect, the present invention provides a method for
treating or controlling an inflammatory disease, condition, or disorder
of the anterior segment. The method comprises administering a composition
comprising a DIGRA, a prodrug thereof, or a pharmaceutically acceptable
salt or ester thereof to an affected eye of a subject in need of such
treatment or control.

[0023]Other features and advantages of the present invention will become
apparent from the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024]FIGS. 1A-1F show the effects of BOL-303242-X and dexamethasone on
the IL-1β-stimulated production of Il-6, IL-7, TGF-α,
TNF-α, VGEF, and MCP-1 in human corneal epithelium cells ("HCECs")
at p<0.05.

[0025]FIG. 2 shows the effects of BOL-303242-X and dexamethasone on the
IL-1β-stimulated production of G-CSF in HCECs at p<0.05.

[0026]FIGS. 3A-1C show the effects of BOL-303242-X and dexamethasone on
the IL-1β-stimulated production of GM-CSF, IL-8, and RANTES in HCECs
at p<0.05.

[0027]In the foregoing figures, "*" denotes comparison to control, and
"**" to IL-1β.

[0028]FIG. 4 shows a comparison of effects of BOL-303242-X (SEGRA) vs. DEX
on myocilin protein in CM of monkey TM cells. Myocilin protein band
densities are represented for a single TM strain in one study. *P<0.05
vs. the vehicle control. † P<0.05 vs. same dose of DEX. Open
bar represents vehicle-treated cells. Two-way ANOVA followed by the
contrast procedure on logarithmically transformed data. Data are
presented as geometric means±SE estimated using the Taylor series
expansion.

[0029]FIG. 5 shows representative quantitative real-time RT-PCR results
for a single strain of monkey TM cells, from a dose-response study
comparing the effects either of BOL-303242-X with DEX, on myocilin mRNA
expression. *P<0.05 vs. vehicle control (open columns). †
P<0.05 for either BOL-303242-X or PA, vs. DEX at the same
concentration tested. Two-way ANOVA followed by the contrast procedure on
logarithmically transformed data (SEGRA vs. DEX) or transformed data
elevated to the power 0.2 (PA vs. DEX). Data are presented as geometric
means±SE estimated using the Taylor series expansion.

DETAILED DESCRIPTION OF THE INVENTION

[0030]As used herein, a dissociated glucocorticoid receptor agonist
("DIGRA") is a compound that is capable of binding to the glucocorticoid
receptor (which is a polypeptide) and, upon binding, is capable of
producing differentiated levels of transrepression and transactivation of
gene expression. A compound that binds to a polypeptide is sometimes
herein referred to as a ligand.

[0031]As used herein, the term "alkyl" or "alkyl group" means a linear- or
branched-chain saturated aliphatic hydrocarbon monovalent group, which
may be unsubstituted or substituted. The group may be partially or
completely substituted with halogen atoms (F, Cl, Br, or I). Non-limiting
examples of alkyl groups include methyl, ethyl, n-propyl,
1-methylethyl(isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl),
and the like. It may be abbreviated as "Alk".

[0032]As used herein, the term "alkenyl" or "alkenyl group" means a
linear- or branched-chain aliphatic hydrocarbon monovalent radical
containing at least one carbon-carbon double bond. This term is
exemplified by groups such as ethenyl, propenyl, n-butenyl, isobutenyl,
3-methylbut-2-enyl, n-pentenyl, heptenyl, octenyl, decenyl, and the like.

[0033]As used herein, the term "alkynyl" or "alkynyl group" means a
linear- or branched-chain aliphatic hydrocarbon monovalent radical
containing at least one carbon-carbon triple bond. This term is
exemplified by groups such as ethynyl, propynyl, n-butynyl, 2-butynyl,
3-methylbutynyl, n-pentynyl, heptynyl, octynyl, decynyl, and the like.

[0034]As used herein, the term "alkylene" or "alkylene group" means a
linear- or branched-chain saturated aliphatic hydrocarbon divalent
radical having the specified number of carbon atoms. This term is
exemplified by groups such as methylene, ethylene, propylene, n-butylene,
and the like, and may alternatively and equivalently be denoted herein as
"-(alkyl)-".

[0035]The term "alkenylene" or "alkenylene group" means a linear- or
branched-chain aliphatic hydrocarbon divalent radical having the
specified number of carbon atoms and at least one carbon-carbon double
bond. This term is exemplified by groups such as ethenylene, propenylene,
n-butenylene, and the like, and may alternatively and equivalently be
denoted herein as "-(alkylenyl)-".

[0036]The term "alkynylene" or "alkynylene group" means a linear- or
branched-chain aliphatic hydrocarbon divalent radical containing at least
one carbon-carbon triple bond. This term is exemplified by groups such as
ethynylene, propynylene, n-butynylene, 2-butynylene, 3-methylbutynylene,
n-pentynylene, heptynylene, octynylene, decynylene, and the like, and may
alternatively and equivalently be denoted herein as "-(alkynyl)-".

[0037]As used herein, the term "aryl" or "aryl group" means an aromatic
carbocyclic monovalent or divalent radical of from 5 to 14 carbon atoms
having a single ring (e.g., phenyl or phenylene), multiple condensed
rings (e.g., naphthyl or anthranyl), or multiple bridged rings (e.g.,
biphenyl). Unless otherwise specified, the aryl ring may be attached at
any suitable carbon atom which results in a stable structure and, if
substituted, may be substituted at any suitable carbon atom which results
in a stable structure. Non-limiting examples of aryl groups include
phenyl, naphthyl, anthryl, phenanthryl, indanyl, indenyl, biphenyl, and
the like. It may be abbreviated as "Ar".

[0039]The term "heterocycle", "heterocycle group", "heterocyclyl",
"heterocyclyl group", "heterocyclic", or "heterocyclic group" means a
stable non-aromatic 5- to 14-membered monocyclic or polycyclic,
monovalent or divalent, ring which may comprise one or more fused or
bridged ring(s), preferably a 5- to 7-membered monocyclic or 7- to
10-membered bicyclic ring, having from one to three heteroatoms in at
least one ring independently selected from nitrogen, oxygen, and sulfur,
wherein any sulfur heteroatoms may optionally be oxidized and any
nitrogen heteroatom may optionally be oxidized or be quaternized. As used
herein, a heterocyclyl group excludes heterocycloalkyl,
heterocycloalkenyl, and heterocycloalkynyl groups. Unless otherwise
specified, the heterocyclyl ring may be attached at any suitable
heteroatom or carbon atom which results in a stable structure and, if
substituted, may be substituted at any suitable heteroatom or carbon atom
which results in a stable structure. Non-limiting examples of
heterocycles include pyrrolinyl, pyrrolidinyl, pyrazolinyl,
pyrazolidinyl, piperidinyl, morpholinyl, thiomorpholinyl, piperazinyl,
tetrahydropyranyl, tetrahydrothiopyranyl, tetrahydrofuranyl,
hexahydropyrimidinyl, hexahydropyridazinyl, and the like.

[0040]The term "cycloalkyl" or "cycloalkyl group" means a stable aliphatic
saturated 3- to 15-membered monocyclic or polycyclic monovalent radical
consisting solely of carbon and hydrogen atoms which may comprise one or
more fused or bridged ring(s), preferably a 5- to 7-membered monocyclic
or 7- to 10-membered bicyclic ring. Unless otherwise specified, the
cycloalkyl ring may be attached at any carbon atom which results in a
stable structure and, if substituted, may be substituted at any suitable
carbon atom which results in a stable structure. Exemplary cycloalkyl
groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,
cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, norbornyl, adamantyl,
tetrahydronaphthyl (tetralin), 1-decalinyl, bicyclo[2.2.2]octanyl,
1-methylcyclopropyl, 2-methylcyclopentyl, 2-methylcyclooctyl, and the
like.

[0041]The term "cycloalkenyl" or "cycloalkenyl group" means a stable
aliphatic 5- to 15-membered monocyclic or polycyclic monovalent radical
having at least one carbon-carbon double bond and consisting solely of
carbon and hydrogen atoms which may comprise one or more fused or bridged
ring(s), preferably a 5- to 7-membered monocyclic or 7- to 10-membered
bicyclic ring. Unless otherwise specified, the cycloalkenyl ring may be
attached at any carbon atom which results in a stable structure and, if
substituted, may be substituted at any suitable carbon atom which results
in a stable structure. Exemplary cycloalkenyl groups include
cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, cyclononenyl,
cyclodecenyl, norbornenyl, 2-methylcyclopentenyl, 2-methylcyclooctenyl,
and the like.

[0042]The term "cycloalkynyl" or "cycloalkynyl group" means a stable
aliphatic 8- to 15-membered monocyclic or polycyclic monovalent radical
having at least one carbon-carbon triple bond and consisting solely of
carbon and hydrogen atoms which may comprise one or more fused or bridged
ring(s), preferably a 8- to 10-membered monocyclic or 12- to 15-membered
bicyclic ring. Unless otherwise specified, the cycloalkynyl ring may be
attached at any carbon atom which results in a stable structure and, if
substituted, may be substituted at any suitable carbon atom which results
in a stable structure. Exemplary cycloalkynyl groups include
cyclooctynyl, cyclononynyl, cyclodecynyl, 2-methylcyclooctynyl, and the
like.

[0043]The term "carbocycle" or "carbocyclic group" means a stable
aliphatic 3- to 15-membered monocyclic or polycyclic monovalent or
divalent radical consisting solely of carbon and hydrogen atoms which may
comprise one or more fused or bridged rings, preferably a 5- to
7-membered monocyclic or 7- to 10-membered bicyclic ring. Unless
otherwise specified, the carbocycle may be attached at any carbon atom
which results in a stable structure and, if substituted, may be
substituted at any suitable carbon atom which results in a stable
structure. The term comprises cycloalkyl (including spiro cycloalkyl),
cycloalkylene, cycloalkenyl, cycloalkenylene, cycloalkynyl, and
cycloalkynylene, and the like.

[0044]The terms "heterocycloalkyl", "heterocycloalkenyl", and
"heterocycloalkynyl" mean cycloalkyl, cycloalkenyl, and cycloalkynyl
group, respectively, having at least a heteroatom in at least one ring,
respectively.

[0045]Glucocorticoids ("GCs") are among the most potent drugs used for the
treatment of allergic and chronic inflammatory diseases or of
inflammation resulting from infections. However, as mentioned above,
long-term treatment with GCs is often associated with numerous adverse
side effects, such as diabetes, osteoporosis, hypertension, glaucoma, or
cataract. These side effects, like other physiological manifestations,
are results of aberrant expression of genes responsible for such
diseases. Research in the last decade has provided important insights
into the molecular basis of GC-mediated actions on the expression of
GC-responsive genes. GCs exert most of their genomic effects by binding
to the cytoplasmic GC receptor ("GR"). The binding of GC to GR induces
the translocation of the GC-GR complex to the cell nucleus where it
modulates gene transcription either by a positive (transactivation) or
negative (transrepression) mode of regulation. There has been growing
evidence that both beneficial and undesirable effects of GC treatment are
the results of undifferentiated levels of expression of these two
mechanisms; in other words, they proceed at similar levels of
effectiveness. Although it has not yet been possible to ascertain the
most critical aspects of action of GCs in chronic inflammatory diseases,
there has been evidence that it is likely that the inhibitory effects of
GCs on cytokine synthesis are of particular importance. GCs inhibit the
transcription, through the transrepression mechanism, of several
cytokines that are relevant in inflammatory diseases, including
IL-1β (interleukin-1β, IL-2, IL-3, IL-6, IL-11, TNF-α
(tumor necrosis factor-α), GM-CSF (granulocyte-macrophage
colony-stimulating factor), and chemokines that attract inflammatory
cells to the site of inflammation, including IL-8, RANTES, MCP-1
(monocyte chemotactic protein-1), MCP-3, MCP-4, MIP-1α
(macrophage-inflammatory protein-1α), and eotaxin. P. J. Barnes,
Clin. Sci., Vol. 94, 557-572 (1998). On the other hand, there is
persuasive evidence that the synthesis of lκB kinases, which are
proteins having inhibitory effects on the NF-κB proinflammatory
transcription factors, is increased by GCs. These proinflammatory
transcription factors regulate the expression of genes that code for many
inflammatory proteins, such as cytokines, inflammatory enzymes, adhesion
molecules, and inflammatory receptors. S. Wissink et al., Mol.
Endocrinol., Vol. 12, No. 3, 354-363 (1998); P. J. Barnes and M. Karin,
New Engl. J. Med., Vol. 336, 1066-1077 (1997). Thus, both the
transrepression and transactivation functions of GCs directed to
different genes produce the beneficial effect of inflammatory inhibition.
On the other hand, steroid-induced diabetes and glaucoma appear to be
produced by the transactivation action of GCs on genes responsible for
these diseases. H. Schacke et al., Pharmacol. Ther., Vol. 96, 23-43
(2002). Thus, while the transactivation of certain genes by GCs produces
beneficial effects, the transactivation of other genes by the same GCs
can produce undesired side effects. Therefore, it is very desirable to
provide pharmaceutical compounds and compositions that produce
differentiated levels of transactivation and transrepression activity on
GC-responsive genes to treat or control inflammatory diseases,
conditions, or disorders, especially chronic inflammation.

[0047]In one aspect, the compounds and compositions of the present
invention cause a lower level of at least an adverse side effect than a
composition comprising at least a prior-art glucocorticoid used to treat
or control the same diseases, conditions, or disorders.

[0048]Ocular inflammatory pathways commence with the triggering of the
arachidonic acid cascade. This cascade is triggered either by mechanical
stimuli (such as the case of unavoidable surgically-inflicted trauma) or
by chemical stimuli (such as foreign substances (e.g., components of
disintegrated pathogenic microorganisms) or allergens). Prostaglandins
are generated in most tissues by activation of the arachidonic acid
pathway. Phospholipids in the damaged cell membrane are the substrate for
phospholipase A to generate arachidonic acid and, in turn, the
cyclooxygenase ("COX") and lipoxygenase enzymes act on arachidonic acid
to produce a family of pro-inflammatory prostaglandins, thromboxanes, and
leukotrienes. These pro-inflammatory compounds recruit more immune cells
(such as macrophages and neutrophils) to the site of injury, which then
produce a greater amount of other pro-inflammatory cytokines and can
further amplify the inflammation.

[0049]Cataract surgery with intraocular lens ("IOL") implantation and
glaucoma filtering microsurgery (trabeculectomy) are among the common
ophthalmic surgical operations. These procedures are usually associated
with some post-operative inflammation. The use of anti-inflammatory
agents post-operatively can rapidly resolve this event to relieve the
patient from pain, discomfort, visual impairment, and to reduce the risk
of further complications (such as the onset of cystoid macular edema).

[0050]Thus, in one aspect, the present invention provides compounds or
compositions for treating or controlling inflammatory diseases,
conditions, or disorders of the anterior segment in a subject, wherein
such inflammatory diseases, conditions, or disorders result from an
infection caused by bacteria, viruses, fungi, protozoans, or combinations
thereof.

[0051]In another aspect, such infection comprises an ocular infection.

[0052]In another aspect, such inflammatory diseases, conditions, or
disorders of the anterior segment result from the physical trauma of
ocular surgery.

[0054]In yet another aspect, the compositions comprise at least a mimetic
of a glucocorticoid for treating or controlling such diseases,
conditions, or disorders.

[0055]In still another aspect, a level of said at least an adverse side
effect is determined in vivo or in vitro. For example, a level of said at
least an adverse side effect is determined in vitro by performing a cell
culture and determining the level of a biomarker associated with said
side effect. Such biomarkers can include proteins (e.g., enzymes),
lipids, sugars, and derivatives thereof that participate in, or are the
products of, the biochemical cascade resulting in the adverse side
effect. Representative in vitro testing methods are further disclosed
hereinbelow.

[0056]In still another aspect, said at least an adverse side effect is
selected from the group consisting of glaucoma, cataract, hypertension,
hyperglycemia, hyperlipidemia (increased levels of triglycerides), and
hypercholesterolemia (increased levels of cholesterol).

[0057]In yet another embodiment, a level of said at least an adverse side
effect is determined at about one day after said composition is first
administered to, and are present in, said subject. In another embodiment,
a level of said at least an adverse side effect is determined about 14
days after said composition is first administered to, and are present in,
said subject. In still another embodiment, a level of said at least an
adverse side effect is determined about 30 days after said composition is
first administered to, and are present in, said subject. Alternatively, a
level of said at least an adverse side effect is determined about 2, 3,
4, 5, or 6 months after said compounds or compositions are first
administered to, and are present in, said subject.

[0058]In another aspect, said at least a prior-art glucocorticoid used to
treat or control the same diseases, conditions, or disorders is
administered to said subject at a dose and a frequency sufficient to
produce an equivalent beneficial effect on said condition to a
composition of the present invention after about the same elapsed time.

[0060]In one aspect, the compounds, compositions, methods of the present
invention provides a lower risk of increased IOP than a composition and
method using a prior-art GC to treat or control the same diseases,
conditions, or disorders.

[0062]In still another aspect, such inflammatory diseases, conditions, or
disorders result from an infection caused by bacteria, viruses, fungi, or
protozoans.

[0063]In yet another aspect, the compositions comprise at least a mimetic
of a glucocorticoid for treating or controlling such diseases,
conditions, or disorders.

[0064]In yet another aspect, a pharmaceutical composition for treating or
controlling an inflammatory disease, condition, or disorder comprises at
least a dissociated glucocorticoid receptor agonist ("DIGRA"), a prodrug,
or a pharmaceutically acceptable salt or ester thereof.

[0066]In still another aspect, such a formulation, system, or device is
applied or provided to the anterior segment of the eye.

[0067]In still another aspect, such a formulation, system, or device is
applied or provided to the posterior segment of the eye.

[0068]In a further aspect, said increased IOP is demonstrated in vitro or
in vivo.

[0069]In yet another aspect, said increased IOP results from an increased
resistance of fluid outflow through the trabecular meshwork.

[0070]In still another aspect, such increased resistance results from
increased expression and accumulation of myocilin in the trabecular
meshwork.

[0071]In another aspect, the present invention provides a method for
treating or controlling an inflammatory disease, condition, or disorder
of the anterior segment. The method comprises administering a composition
comprising a DIGRA, a prodrug thereof, or a pharmaceutically acceptable
salt or ester thereof to an affected eye of a subject in need of such
treatment or control.

[0072]In still another aspect, said at least a DIGRA has Formula I.

##STR00001##

wherein A and Q are independently selected from the group consisting of
unsubstituted and substituted aryl and heteroaryl groups, unsubstituted
and substituted cycloalkyl and heterocycloalkyl groups, unsubstituted and
substituted cycloalkenyl and heterocycloalkenyl groups, unsubstituted and
substituted cycloalkynyl and heterocycloalkynyl groups, and unsubstituted
and substituted heterocyclic groups; R1 and R2 are
independently selected from the group consisting of hydrogen,
unsubstituted C1-C15 (alternatively, C1-C10, or
C1-C5, or C1-C3) linear or branched alkyl groups,
substituted C1-C15 (alternatively, C1-C10, or
C1-C5, or C1-C3) linear or branched alkyl groups,
unsubstituted C3-C15 cycloalkyl groups, and substituted
C3-C15 (alternatively, C3-C6, or C3-C5)
cycloalkyl groups; R3 is selected from the group consisting of
hydrogen, unsubstituted C1-C15 (alternatively,
C1-C10, or C1-C5, or C1-C3) linear or
branched alkyl groups, substituted C1-C15 (alternatively,
C1-C10, or C1-C5, or C1-C3) linear or
branched alkyl groups, unsubstituted C3-C15 (alternatively,
C3-C6, or C3-C5) cycloalkyl and heterocycloalkyl
groups, substituted C3-C15 (alternatively, C3-C6, or
C3-C5) cycloalkyl and heterocycloalkyl groups, aryl groups,
heteroaryl groups, and heterocyclylic groups; B comprises a carbonyl,
amino, divalent hydrocarbon, or heterohydrocarbon group; E is hydroxy or
amino group; and D is absent or comprises a carbonyl group, --NH--, or
--NR'--, wherein R' comprises an unsubstituted or substituted
C1-C15 (alternatively, C1-C10), or C1-C5,
or C1-C3) linear or branched alkyl group; and wherein R1
and R2 together may form an unsubstituted or substituted
C3-C15 cycloalkyl group.

[0073]In one embodiment, B can comprise one or more unsaturated
carbon-carbon bonds.

[0075]In still another embodiment, A and Q are independently selected from
the group consisting of aryl and heteroaryl groups substituted with at
least a halogen atom, cyano group, hydroxy group, or C1-C10
alkoxy group (alternatively, C1-C5 alkoxy group, or
C1-C3 alkoxy group); R1, R2, and R3 are
independently selected from the group consisting of unsubstituted and
substituted C1-C5 alkyl groups (preferably, C1-C3
alkyl groups); B is a C1-C5 alkylene group (alternatively,
C1-C3 alkyl groups); D is the --NH-- or --NR'-- group, wherein
R' is a C1-C5 alkyl group (preferably, C1-C3 alkyl
group); and E is the hydroxy group.

[0076]In yet another embodiment, A comprises a dihydrobenzofuranyl group
substituted with a halogen atom; Q comprises a quinolinyl or
isoquinolinyl group substituted with a C1-C10 alkyl group;
R1 and R2 are independently selected from the group consisting
of unsubstituted and substituted C1-C5 alkyl groups
(preferably, C1-C3 alkyl groups); B is a C1-C3
alkylene group; D is the --NH-- group; E is the hydroxy group; and
R3 comprises a completely halogenated C1-C10 alkyl group
(preferably, completely halogenated C1-C5 alkyl group; more
preferably, completely halogenated C1-C3 alkyl group).

[0077]In still another embodiment, A comprises a dihydrobenzofuranyl group
substituted with a fluorine atom; Q comprises a quinolinyl or
isoquinolinyl group substituted with a methyl group; R1 and R2
are independently selected from the group consisting of unsubstituted and
substituted C1-C5 alkyl groups; B is a C1-C3 alkylene
group; D is the --NH-- group; E is the hydroxy group; and R3
comprises a trifluoromethyl group.

[0078]In a further embodiment, said at least a DIGRA has Formula II or
III.

[0079]In still another embodiment, said at least a DIGRA has Formula IV.

##STR00003##

[0080]Methods for preparing compounds of Formula I, II, III, or IV are
disclosed, for example, in U.S. Pat. Nos. 6,897,224; 6,903,215;
6,960,581, which are incorporated herein by reference in their entirety.
Still other methods for preparing such compounds also can be found in
U.S. Patent Application Publication 2006/0116396, which is incorporated
herein by reference, or PCT Patent Application WO 2006/050998 A1.

[0082]Other compounds that can function as DIGRAs and methods for their
manufacture are disclosed, for example, in U.S. Patent Application
Publications 2004/0029932, 2004/0162321, 2004/0224992, 2005/0059714,
2005/0176706, 2005/0203128, 2005/0234091, 2005/0282881, 2006/0014787,
2006/0030561, 2006/0116396, 2006/0189646, and 2006/0189647, all of which
are incorporated herein by reference in their entirety.

[0083]In another aspect, the present invention provides an ophthalmic
pharmaceutical composition for treating or controlling an
anterior-segment infection and its inflammatory sequalae. In one
embodiment, such inflammatory sequalae comprise acute inflammation. In
another embodiment, such inflammatory sequalae comprise chronic
inflammation of the anterior segment. The ophthalmic pharmaceutical
composition comprises a DIGRA, a prodrug thereof, or a pharmaceutically
acceptable salt or ester thereof.

[0084]In another aspect, the composition further comprises an
anti-infective agent.

[0085]In still another aspect, the pharmaceutical composition further
comprises a pharmaceutically acceptable carrier.

[0086]The concentration of a DIGRA, a prodrug thereof, or a
pharmaceutically acceptable salt or ester thereof in such an ophthalmic
composition can be in the range from about 0.0001 to about 1000 mg/ml
(or, alternatively, from about 0.001 to about 500 mg/ml, or from about
0.001 to about 300 mg/ml, or from about 0.001 to about 250 mg/ml, or from
about 0.001 to about 100 mg/ml, or from about 0.001 to about 50 mg/ml, or
from about 0.01 to about 300 mg/ml, or from about 0.01 to about 250
mg/ml, or from about 0.01 to about 100 mg/ml, or from about 0.1 to about
100 mg/ml, or from about 0.1 to about 50 mg/ml).

[0087]In one embodiment, a composition of the present invention is in a
form of a suspension, dispersion, gel, or ointment. In another
embodiment, the suspension or dispersion is based on an aqueous solution.
For example, a composition of the present invention can comprise sterile
saline solution. In still another embodiment, micrometer- or
nanometer-sized particles of a DIGRA, or prodrug thereof, or a
pharmaceutically acceptable salt or ester thereof and can be coated with
a physiologically acceptable surfactant (non-limiting examples are
disclosed below), then the coated particles are dispersed in a liquid
medium. The coating can keep the particles in a suspension. Such a liquid
medium can be selected to produce a sustained-release suspension. For
example, the liquid medium can be one that is sparingly soluble in the
ocular environment into which the suspension is administered.

[0088]An anti-infective agent suitable for a composition of the present
invention is selected from the group consisting of antibacterial,
antiviral, antifungal, antiprotozoal, and combinations thereof.

[0096]The concentration of an anti-infective agent in such an ophthalmic
composition can be in the range from about 0.0001 to about 1000 mg/ml
(or, alternatively, from about 0.001 to about 500 mg/ml, or from about
0.001 to about 300 mg/ml, or from about 0.001 to about 250 mg/ml, or from
about 0.001 to about 100 mg/ml, or from about 0.001 to about 50 mg/ml, or
from about 0.01 to about 300 mg/ml, or from about 0.01 to about 250
mg/ml, or from about 0.01 to about 100 mg/ml, or from about 0.1 to about
100 mg/ml, or from about 0.1 to about 50 mg/ml).

[0097]In another aspect, a composition of the present invention can
further comprise a non-ionic surfactant, such as polysorbates (such as
polysorbate 80 (polyoxyethylene sorbitan monooleate), polysorbate 60
(polyoxyethylene sorbitan monostearate), polysorbate 20 (polyoxyethylene
sorbitan monolaurate), commonly known by their trade names of Tween®
80, Tween® 60, Tween® 20), poloxamers (synthetic block polymers
of ethylene oxide and propylene oxide, such as those commonly known by
their trade names of Pluronic®; e.g., Plutonic® F127 or
Pluronic® F108)), or poloxamines (synthetic block polymers of
ethylene oxide and propylene oxide attached to ethylene diamine, such as
those commonly known by their trade names of Tetronic®; e.g.,
Tetronic® 1508 or Tetronic® 908, etc., other nonionic surfactants
such as Brij®, Myrj®, and long chain fatty alcohols (i.e., oleyl
alcohol, stearyl alcohol, myristyl alcohol, docosohexanoyl alcohol, etc.)
with carbon chains having about 12 or more carbon atoms (e.g., such as
from about 12 to about 24 carbon atoms). Such compounds are delineated in
Martindale, 34th ed., pp. 1411-1416 (Martindale, "The Complete Drug
Reference," S. C. Sweetman (Ed.), Pharmaceutical Press, London, 2005) and
in Remington, "The Science and Practice of Pharmacy," 21st Ed., p.
291 and the contents of chapter 22, Lippincott Williams & Wilkins, New
York, 2006); the contents of these sections are incorporated herein by
reference. The concentration of a non-ionic surfactant, when present, in
a composition of the present invention can be in the range from about
0.001 to about 5 weight percent (or alternatively, from about 0.01 to
about 4, or from about 0.01 to about 2, or from about 0.01 to about 1, or
from about 0.01 to about 0.5 weight percent).

[0098]In addition, a composition of the present invention can include
additives such as buffers, diluents, carriers, adjuvants, or other
excipients. Any pharmacologically acceptable buffer suitable for
application to the eye may be used. Other agents may be employed in the
composition for a variety of purposes. For example, buffering agents,
preservatives, co-solvents, oils, humectants, emollients, stabilizers, or
antioxidants may be employed. Water-soluble preservatives which may be
employed include sodium bisulfite, sodium bisulfate, sodium thiosulfate,
benzalkonium chloride, chlorobutanol, thimerosal, ethyl alcohol,
methylparaben, polyvinyl alcohol, benzyl alcohol, and phenylethyl
alcohol. These agents may be present in individual amounts of from about
0.001 to about 5% by weight (preferably, about 0.01% to about 2% by
weight). Suitable water-soluble buffering agents that may be employed are
sodium carbonate, sodium borate, sodium phosphate, sodium acetate, sodium
bicarbonate, etc., as approved by the United States Food and Drug
Administration ("US FDA") for the desired route of administration. These
agents may be present in amounts sufficient to maintain a pH of the
system of between about 2 and about 11. As such, the buffering agent may
be as much as about 5% on a weight to weight basis of the total
composition. Electrolytes such as, but not limited to, sodium chloride
and potassium chloride may also be included in the formulation.

[0099]In one aspect, the pH of the composition is in the range from about
4 to about 11. Alternatively, the pH of the composition is in the range
from about 5 to about 9, from about 6 to about 9, or from about 6.5 to
about 8. In another aspect, the composition comprises a buffer having a
pH in one of said pH ranges.

[0100]In another aspect, the composition has a pH of about 7.
Alternatively, the composition has a pH in a range from about 7 to about
7.5.

[0101]In still another aspect, the composition has a pH of about 7.4.

[0102]In yet another aspect, a composition also can comprise a
viscosity-modifying compound designed to facilitate the administration of
the composition into the subject or to promote the bioavailability in the
subject. In still another aspect, the viscosity-modifying compound may be
chosen so that the composition is not readily dispersed after being
administered into the vistreous. Such compounds may enhance the viscosity
of the composition, and include, but are not limited to: monomeric
polyols, such as, glycerol, propylene glycol, ethylene glycol; polymeric
polyols, such as, polyethylene glycol; various polymers of the cellulose
family, such as hydroxypropylmethyl cellulose ("HPMC"), carboxymethyl
cellulose ("CMC") sodium, hydroxypropyl cellulose ("HPC");
polysaccharides, such as hyaluronic acid and its salts, chondroitin
sulfate and its salts, dextrans, such as, dextran 70; water soluble
proteins, such as gelatin; vinyl polymers, such as, polyvinyl alcohol,
polyvinylpyrrolidone, povidone; carbomers, such as carbomer 934P,
carbomer 941, carbomer 940, or carbomer 974P; and acrylic acid polymers.
In general, a desired viscosity can be in the range from about 1 to about
400 centipoises ("cps") or mPas.

[0103]In yet another aspect, the present invention provides a composition
for treating or controlling an ophthalmic (anterior and or posterior
segment) inflammatory disease, condition, or disorder. In one embodiment,
the composition comprises: (a) at least a DIGRA, a prodrug thereof, or a
pharmaceutically acceptable salt or ester thereof and (b) an
anti-inflammatory agent other than said DIGRA, prodrug thereof, and
pharmaceutically acceptable salt or ester thereof. In another embodiment,
the anti-inflammatory agent is not a GC.

[0104]In still another aspect, such an anti-inflammatory agent comprises a
compound that inhibits or blocks a cyclooxygenase inflammatory pathway, a
lipoxygenase inflammatory pathway, or both.

[0105]In still another aspect, such an anti-inflammatory agent comprises a
compound that inhibits or blocks production of a prostaglandin,
thromboxane, or leukotriene.

[0106]In yet another aspect, the present invention provides a composition
for treating or controlling an ophthalmic (anterior and/or posterior
segment) inflammatory disease, condition, or disorder. In one embodiment,
the composition comprises: (a) at least a DIGRA, a prodrug thereof, or a
pharmaceutically acceptable salt or ester thereof (b) an anti-infective
agent; and (c) an anti-inflammatory agent other than said DIGRA, prodrug
thereof, and pharmaceutically acceptable salt or ester thereof. The
DIGRA, anti-infective agent, and anti-inflammatory agent other than said
DIGRA, prodrug thereof, and pharmaceutically acceptable salt or ester
thereof are present in amounts effective to treat or control the disease,
condition, or disorder. In one embodiment, such an anti-inflammatory
agent is selected from the group consisting of non-steroidal
anti-inflammatory drugs ("NSAIDs"); peroxisome proliferator-activated
receptor ("PPAR") ligands, such as PPARα, PPARδ, or
PPARγ ligands; combinations thereof; and mixtures thereof.

[0108]In certain embodiments, said anti-inflammatory agent other than said
DIGRA, prodrug thereof, and pharmaceutically acceptable salt or ester
thereof is selected from the group consisting of flurbiprofen, suprofen,
bromfenac, diclofenac, indomethacin, ketorolac, salts thereof, and
combinations thereof.

[0109]In another aspect of the present invention, an anti-inflammatory
agent is a PPAR-binding molecule. In one embodiment, such a PPAR-binding
molecule is a PPARα-, PPARδ-, or PPARγ-binding
molecule. In another embodiment, such a PPAR-binding molecule is a
PPARα, PPARδ, or PPARγ agonist. Such a PPAR ligand
binds to and activates PPAR to modulate the expression of genes
containing the appropriate peroxisome proliferator response element in
its promoter region.

[0110]PPARγ agonists can inhibit the production of TNF-α and
other inflammatory cytokines by human macrophages (C-Y. Jiang et al.,
Nature, Vol. 391, 82-86 (1998)) and T lymphocytes (A. E. Giorgini et al.,
Horm. Metab. Res. Vol. 31, 1-4 (1999)). More recently, the natural
PPARγ agonist 15-deoxy-A-12,14-prostaglandin J2 (or
"15-deoxy-Δ-12,14-PG J2"), has been shown to inhibit
neovascularization and angiogenesis (X. Xin et al., J. Biol. Chem. Vol.
274:9116-9121 (1999)) in the rat cornea. Spiegelman et al., in U.S. Pat.
No. 6,242,196, disclose methods for inhibiting proliferation of
PPARγ-responsive hyperproliferative cells by using PPARγ
agonists; numerous synthetic PPARγ agonists are disclosed by
Spiegelman et al., as well as methods for diagnosing
PPARγ-responsive hyperproliferative cells. All documents referred
to herein are incorporated by reference. PPARs are differentially
expressed in diseased versus normal cells. PPARγ is expressed to
different degrees in the various tissues of the eye, such as some layers
of the retina and the cornea, the choriocapillaris, uveal tract,
conjunctival epidermis, and intraocular muscles (see, e.g., U.S. Pat. No.
6,316,465).

[0112]Non-limiting examples of PPAR-α agonists include the fibrates,
such as fenofibrate and gemfibrozil. A non-limiting example of
PPAR-δ agonist is GW501516 (available from Axxora LLC, San Diego,
Calif. or EMD Biosciences, Inc., San Diego, Calif.).

[0113]The concentration of any foregoing additional active ingredient in
such an ophthalmic composition can be in the range from about 0.0001 to
about 1000 mg/ml (or, alternatively, from about 0.001 to about 500 mg/ml,
or from about 0.001 to about 300 mg/ml, or from about 0.001 to about 250
mg/ml, or from about 0.001 to about 100 mg/ml, or from about 0.001 to
about 50 mg/ml, or from about 0.01 to about 300 mg/ml, or from about 0.01
to about 250 mg/ml, or from about 0.01 to about 100 mg/ml, or from about
0.1 to about 100 mg/ml, or from about 0.1 to about 50 mg/ml).

[0114]In still another aspect, a method for preparing a composition of the
present invention comprises combining: (a) at least a DIGRA, a prodrug
thereof, or a pharmaceutically acceptable salt or ester thereof (b) a
pharmaceutically acceptable carrier; and (c) a material selected from the
group consisting of (i) an anti-infective agent, (ii) an
anti-inflammatory agent other than said DIGRA, prodrug thereof, and
pharmaceutically acceptable salt or ester thereof; and (iii) combinations
thereof. In one embodiment, such a carrier can be a sterile saline
solution or a physiologically acceptable buffer. In another embodiment,
such a carrier comprises a hydrophobic medium, such as a pharmaceutically
acceptable oil. In still another embodiment, such as carrier comprises an
emulsion of a hydrophobic material and water.

[0115]Physiologically acceptable buffers include, but are not limited to,
a phosphate buffer or a Tris-HCl buffer (comprising
tris(hydroxymethyl)aminomethane and HCl). For example, a Tris-HCl buffer
having pH of 7.4 comprises 3 g/l of tris(hydroxymethyl)aminomethane and
0.76 g/l of HCl. In yet another aspect, the buffer is 10× phosphate
buffer saline ("PBS") or 5×PBS solution.

[0116]Other buffers also may be found suitable or desirable in some
circumstances, such as buffers based on HEPES
(N-{2-hydroxyethyl}peperazine-N'-{2-ethanesulfonic acid}) having pKa
of 7.5 at 25° C. and pH in the range of about 6.8-8.2; BES
(N,N-bis{2-hydroxyethyl}2-aminoethanesulfonic acid) having pKa of
7.1 at 25° C. and pH in the range of about 6.4-7.8; MOPS
(3-{N-morpholino}propanesulfonic acid) having pKa of 7.2 at
25° C. and pH in the range of about 6.5-7.9; TES
(N-tris{hydroxymethyl}-methyl-2-aminoethanesulfonic acid) having pKa
of 7.4 at 25° C. and pH in the range of about 6.8-8.2; MOBS
(4-{N-morpholino}butanesulfonic acid) having pKa of 7.6 at
25° C. and pH in the range of about 6.9-8.3; DIPSO
(3-(N,N-bis{2-hydroxyethyl}amino)-2-hydroxypropane)) having pKa of
7.52 at 25° C. and pH in the range of about 7-8.2; TAPSO
(2-hydroxy-3{tris(hydroxymethyl)methylamino}-1-propanesulfonic acid))
having pKa of 7.61 at 25° C. and pH in the range of about
7-8.2; TAPS
({(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)amino}-1-propanesulfonic acid))
having pKa of 8.4 at 25° C. and pH in the range of about
7.7-9.1; TABS (N-tris(hydroxymethyl)methyl-4-aminobutanesulfonic acid)
having pKa of 8.9 at 25° C. and pH in the range of about
8.2-9.6; AMPSO
(N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid))
having pKa of 9.0 at 25° C. and pH in the range of about
8.3-9.7; CHES (2-cyclohexylamino)ethanesulfonic acid) having pKa of
9.5 at 25° C. and pH in the range of about 8.6-10.0; CAPSO
(3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid) having pKa of
9.6 at 25° C. and pH in the range of about 8.9-10.3; or CAPS
(3-(cyclohexylamino)-1-propane sulfonic acid) having pKa of 10.4 at
25° C. and pH in the range of about 9.7-11.1.

[0117]In certain embodiments, a composition of the present invention is
formulated in a buffer having an acidic pH, such as from about 4 to about
6.8, or alternatively, from about 5 to about 6.8. In such embodiments,
the buffer capacity of the composition desirably allows the composition
to come rapidly to a physiological pH after being administered into the
patient.

[0118]It should be understood that the proportions of the various
components or mixtures in the following examples may be adjusted for the
appropriate circumstances.

Example 1

[0119]Two mixtures I and II are made separately by mixing the ingredients
listed in Table 1. Five parts (by weight) of mixture I are mixed with
twenty parts (by weight) of mixture II for 15 minutes or more. The pH of
the combined mixture is adjusted to 6.2-6.4 using 1 N NaOH or 1 N HCl
solution to yield a composition of the present invention.

[0120]Two mixtures I and II are made separately by mixing the ingredients
listed in Table 2. Five parts (by weight) of mixture I are mixed with
twenty parts (by weight) of mixture II for 15 minutes or more. The pH of
the combined mixture is adjusted to 6.2-6.4 using 1 N NaOH or 1 N HCl
solution to yield a composition of the present invention.

[0121]Two mixtures I and II are made separately by mixing the ingredients
listed in Table 3. Five parts (by weight) of mixture I are mixed with
twenty parts (by weight) of mixture II for 15 minutes or more. The pH of
the combined mixture is adjusted to 6.2-6.4 using 1 N NaOH or 1 N HCl
solution to yield a composition of the present invention.

[0122]Two mixtures I and II are made separately by mixing the ingredients
listed in Table 4. Five parts (by weight) of mixture I are mixed with
twenty parts (by weight) of mixture II for 15 minutes or more. The pH of
the combined mixture is adjusted to 6.2-7.5 using 1 N NaOH or 1 N HCl
solution to yield a composition of the present invention.

[0123]The ingredients listed in Table 5 are mixed together for at least 15
minutes. The pH of the mixture is adjusted to 6.2-7.5 using 1 N NaOH or 1
N HCl solution to yield a composition of the present invention.

[0124]The ingredients listed in Table 6 are mixed together for at least 15
minutes. The pH of the mixture is adjusted to 7-7.5 using 1 N NaOH or 1 N
HCl solution to yield a composition of the present invention.

[0125]The ingredients listed in Table 7 are mixed together for at least 15
minutes. The pH of the mixture is adjusted to 6.5-7.8 using 1 N NaOH or 1
N HCl solution to yield a composition of the present invention.

[0126]The ingredients listed in Table 8 are mixed together for at least 15
minutes. The pH of the mixture is adjusted to 6.2-7.4 using 1 N NaOH or 1
N HCl solution to yield a composition of the present invention.

[0127]The ingredients listed in Table 9 are mixed together for at least 15
minutes. The pH of the mixture is adjusted to 6.2-6.8 using 1 N NaOH or 1
N HCl solution to yield a composition of the present invention.

[0128]The ingredients listed in Table 10 are mixed together for at least
15 minutes. The pH of the mixture is adjusted to 6.2-6.8 using 1 N NaOH
or 1 N HCl solution to yield a composition of the present invention.

[0129]The ingredients listed in Table 11 are mixed together for at least
15 minutes. The pH of the mixture is adjusted to 6.2-7 using 1 N NaOH or
1 N HCl solution to yield a composition of the present invention.

[0130]In another aspect, a DIGRA, a prodrug thereof, or a pharmaceutically
acceptable salt or ester thereof is incorporated into a formulation for
topical administration or periocular injection to a portion of the
anterior segment. An injectable formulation can desirably comprise a
carrier that provides a sustained-release of the active ingredients, such
as for a period longer than about 1 week (or longer than about 1, 2, 3,
4, 5, or 6 months). In certain embodiments, the sustained-release
formulation desirably comprises a carrier that is insoluble or only
sparingly soluble in the anterior-segment environment. Such a carrier can
be an oil-based liquid, emulsion, gel, or semisolid. Non-limiting
examples of oil-based liquids include castor oil, peanut oil, olive oil,
coconut oil, sesame oil, cottonseed oil, corn oil, sunflower oil,
fish-liver oil, arachis oil, and liquid paraffin.

[0131]In another embodiment, the formulation further comprises a material
selected from the group consisting of: (i) anti-infective agents; (ii)
anti-inflammatory agents other than said DIGRA, prodrug thereof,
pharmaceutically acceptable salts, and pharmaceutically acceptable esters
thereof; and (iii) a combination thereof.

[0132]In one embodiment, a compound or composition of the present
invention can be injected with a fine-gauge needle, such as 25-35 gauge.
Typically, an amount from about 25 μl to about 100 μl of a
composition comprising a DIGRA, a prodrug thereof, or a pharmaceutically
acceptable salt or ester thereof is administered into a patient. A
concentration of such DIGRA, prodrug thereof, or pharmaceutically
acceptable salt or ester thereof is selected from the ranges disclosed
above.

[0133]In still another aspect, a DIGRA, a prodrug thereof, or a
pharmaceutically acceptable salt or ester thereof is incorporated into an
ophthalmic device that comprises a biodegradable material, and the device
is implanted into an anterior-segment tissue of a subject to provide a
long-term (e.g., longer than about 1 week, or longer than about 1, 2, 3,
4, 5, or 6 months) treatment or control of an anterior-segment
inflammatory disease, condition, or disorder. Such a device may be
implanted by a skilled physician in the subject's ocular or periocular
tissue.

[0134]In still another aspect, a method for treating or controlling an
ocular inflammatory disease, condition, or disorder comprises: (a)
providing a composition comprising a DIGRA, a prodrug thereof, or a
pharmaceutically acceptable salt or ester thereof; and (b) administering
to a subject an amount of the composition at a frequency sufficient to
treat or control said ocular disease, condition, or disorder in a
subject, wherein said method results in a lower risk of increased IOP in
said subject than a method using a prior-art GC. In one embodiment, said
prior-art GC is dexamethasone. In another embodiment, said prior-art GC
is prednisolone acetate.

[0135]In still another aspect, a method for treating or controlling a
post-operative inflammation of the anterior segment comprises: (a)
providing a composition comprising a DIGRA, a prodrug thereof, or a
pharmaceutically acceptable salt or ester thereof; and (b) administering
to a subject an amount of the composition at a frequency sufficient to
treat or control said post-operative inflammation, wherein said method
results in a lower risk of increased IOP in said subject than a method
using a prior-art GC. In one embodiment, said prior-art GC is
dexamethasone. In another embodiment, said prior-art GC is prednisolone
acetate.

[0136]In still another aspect, a method for treating or controlling an
anterior-segment inflammatory disease, condition, or disorder comprises:
(a) providing a composition comprising a DIGRA, a prodrug thereof, or a
pharmaceutically acceptable salt or ester thereof; and (b) administering
to a subject an amount of the composition at a frequency sufficient to
treat or control an anterior-segment disease, condition, or disorder in a
subject, wherein said method results in a lower risk of increased IOP in
said subject than a method using a prior-art GC. In one embodiment, said
prior-art GC is dexamethasone. In another embodiment, said prior-art GC
is prednisolone acetate.

[0137]In still another aspect, a method for treating or controlling a
post-operative inflammation of the anterior segment comprises: (a)
providing a composition comprising a DIGRA, a prodrug thereof, or a
pharmaceutically acceptable salt or ester thereof; and (b) administering
to a subject an amount of the composition at a frequency sufficient to
treat or control said post-operative inflammation, wherein said method
results in a lower risk of increased IOP in said subject than a method
using a prior-art GC. In one embodiment, said prior-art GC is
dexamethasone. In another embodiment, said prior-art GC is prednisolone
acetate.

[0138]In still another aspect, a method for treating or controlling an
anterior-segment inflammatory disease, condition, or disorder comprises:
(a) providing a composition comprising: (i) a DIGRA, a prodrug thereof,
or a pharmaceutically acceptable salt or ester thereof; (ii) an
anti-inflammatory agent other than said DIGRA, prodrug thereof, and
pharmaceutically acceptable salt or ester thereof; and (iii) an
anti-infective agent; and (b) administering to a subject an amount of the
composition at a frequency sufficient to treat or control an
anterior-segment disease, condition, or disorder in a subject, wherein
said method results in a lower risk of increased IOP in said subject than
a method using a prior-art GC. In one embodiment, said prior-art GC is
dexamethasone. In another embodiment, said prior-art GC is prednisolone
acetate.

[0139]In still another aspect, a method for treating or controlling a
post-operative inflammation of the anterior segment comprises: (a)
providing a composition comprising: (i) a DIGRA, a prodrug thereof, or a
pharmaceutically acceptable salt or ester thereof; (ii) an
anti-inflammatory agent other than said DIGRA, prodrug thereof, and
pharmaceutically acceptable salt or ester thereof; and (iii) an
anti-infective agent; and (b) administering to a subject an amount of the
composition at a frequency sufficient to treat or control said
post-operative inflammation, wherein said method results in a lower risk
of increased IOP in said subject than a method using a prior-art GC. In
one embodiment, said prior-art GC is dexamethasone. In another
embodiment, said prior-art GC is prednisolone acetate.

[0140]In certain embodiments, the DIGRA is selected from among those
disclosed above.

[0141]In other embodiments, the anti-inflammatory agent is selected from
among those disclosed above. In some embodiments, the anti-inflammatory
agent is selected from the group consisting of flurbiprofen, suprofen,
bromfenac, diclofenac, indomethacin, ketorolac, salts thereof, and
combinations thereof.

[0142]In another embodiment, such inflammation is a long-term
inflammation. In still another embodiment, such inflammation requires at
least two weeks for resolution, if untreated.

[0143]In still another embodiment, such inflammatory anterior-segment
disease, condition, or disorder results from ophthalmic infection that is
caused by a virus, bacteria, fungus, or protozoa.

[0144]In another aspect, a composition of the present invention is
administered periocularly or in the anterior chamber. In still another
aspect, a composition of the present invention is incorporated into an
ophthalmic implant system or device, and the implant system or device is
surgically implanted periocularly or in a tissue adjacent to the anterior
portion of the eye of the patient for the sustained release of the active
ingredient or ingredients. A typical implant system or device suitable
for use in a method of the present invention comprises a biodegradable
matrix with the active ingredient or ingredients impregnated or dispersed
therein. Non-limiting examples of ophthalmic implant systems or devices
for the sustained-release of an active ingredient are disclosed in U.S.
Pat. Nos. 5,378,475; 5,773,019; 5,902,598; 6,001,386; 6,051,576; and
6,726,918; which are incorporated herein by reference.

[0145]In yet another aspect, a composition of the present invention is
administered once a week, once a month, once a year, twice a year, four
times a year, or at a suitable frequency that is determined to be
appropriate for treating or controlling an anterior-segment inflammatory
disease, condition, or disorder.

[0146]Comparison of Glucocorticoids and DIGRAS

[0147]One of the most frequent undesirable actions of a glucocorticoid
therapy is steroid diabetes. The reason for this undesirable condition is
the stimulation of gluconeogenesis in the liver by the induction of the
transcription of hepatic enzymes involved in gluconeogenesis and
metabolism of free amino acids that are produced from the degradation of
proteins (catabolic action of glucocorticoids). A key enzyme of the
catabolic metabolism in the liver is the tyrosine aminotransferase
("TAT"). The activity of this enzyme can be determined photometrically
from cell cultures of treated rat hepatoma cells. Thus, the
gluconeogenesis by a glucocorticoid can be compared to that of a DIGRA by
measuring the activity of this enzyme. For example, in one procedure, the
cells are treated for 24 hours with the test substance (a DIGRA or
glucocorticoid), and then the TAT activity is measured. The TAT
activities for the selected DIGRA and glucocorticoid are then compared.
Other hepatic enzymes can be used in place of TAT, such as
phosphoenolpyruvate carboxykinase, glucose-6-phosphatase, or
fructose-2,6-biphosphatase. Alternatively, the levels of blood glucose in
an animal model may be measured directly and compared for individual
subjects that are treated with a glucocorticoid for a selected condition
and those that are treated with a DIGRA for the same condition.

[0148]Another undesirable result of glucocorticoid therapy is GC-induced
cataract. The cataractogenic potential of a compound or composition may
be determined by quantifying the effect of the compound or composition on
the flux of potassium ions through the membrane of lens cells (such as
mammalian lens epithelial cells) in vitro. Such an ion flux may be
determined by, for example, electrophysiological techniques or ion-flux
imaging techniques (such as with the use of fluorescent dyes). An
exemplary in-vitro method for determining the cataractogenic potential of
a compound or composition is disclosed in U.S. Patent Application
Publication 2004/0219512, which is incorporated herein by reference.

[0149]Still another undesirable result of glucocorticoid therapy is
hypertension. Blood pressure of similarly matched subjects treated with
glucocorticoid and DIGRA for an inflammatory condition may be measured
directly and compared.

[0150]Yet another undesirable result of glucocorticoid therapy is
increased IOP in the subject. IOP of similarly matched subjects treated
with glucocorticoid and DIGRA for a condition may be measured directly
and compared.

TESTING: Comparison of the DIGRA Having Formula IV With Two
Corticosteroids and One NSA/D in Treating Anterior-Segment Inflammation

1. Introduction

[0151]Inflammatory processes are multidimensional in origin, and are
characterized by complex cellular and molecular events involving numerous
components all of which have not been identified. Prostaglandins are
among these mediators and play an important role in certain forms of
ocular inflammation. Paracentesis of the anterior chamber in the rabbit
eye induces inflammatory reaction due to the disruption of the
blood-aqueous barrier ("BAB"), which is mediated, at least in part, by
prostaglandin E2 [References 1-3 below]. Intraocular or topical
administration of PGE2 disrupts the BAB. [Reference 4, below] The
treatment schedule adopted in this study was similar to the clinical
NSAIDs (Ocufen) treatment schedule used by surgeons for patients before
cataract surgery. We investigated a dissociated glucocorticoid receptor
agonist ("BOL-303242-X", compound having Formula IV above) at different
doses on rabbit paracentesis model evaluating aqueous biomarkers levels,
and iris-ciliary body MPO activity in comparison with vehicle,
dexamethasone, loteprednol and flurbiprofen.

[0165]Justification: The rabbit is a standard non-rodent species used in
pharmacodynamic studies. The number of animals used in this study is, in
judgment of the investigators involved, the minimum number necessary to
properly perform this type of study and it is consistent with world wide
regulatory guidelines.

[0166]Acclimation/Quarantine: Following arrival, a member of the
veterinary staff assessed animals as to their general health. Seven days
elapsed between animal receipt and the start of experiment in order to
acclimate animals to the laboratory environment and to observe them for
the development of infection disease.

[0167]Animal Husbandry: All the animals were housed in a cleaned and
disinfected room, with a constant temperature (22±1° C.),
humidity (relative, 30%) and under a constant light-dark cycle (light on
between 8.00 and 20.00). Commercial food and tap water were available ad
libitum. Their body weights were measured just before the experiment
(Table T-1). All the animals had a body weight inside the central part of
the body weight distribution curve (10%). Four rabbits were replaced with
animals of similar age and weight from the same vendor because three of
them showed signs of ocular inflammation and one was dead upon arrival.

[0168]Animals Welfare Provisions: All experiments were carried out
according to the ARVO (Association for Research in Vision and
Ophthalmology) guidelines on the use of animals in research. No
alternative test system exists which have been adequately validated to
permit replacement of the use of live animals in this study. Every effort
has been made to obtain the maximum amount of information while reducing
to a minimum the number of animals required for this study. To the best
of our knowledge, this study is not unnecessary or duplicative. The study
protocol was reviewed and approved by the Institutional Animal Care and
Use Committee (IACUC) of the University of Catania and complies with the
acceptable standards of animal welfare care.

2.3 Experimental Preparations

2.3.1 Study Design and Randomization

[0169]Twenty-eight rabbits were randomly allocated into 7 groups (4
animals/each) as shown in the table below.

[0181]The solution was prepared freshly. Ten microliters of H2O2
(30 wt. %) were diluted to 1 ml with water (solution A). 7.5 mg
o-dianisidine 2HCl was dissolved in 45 ml of phosphate buffer and 75
μl of solution A were added.

2.4 Experimental Protocols

2.4.1 Animals Treatment and Sample Collection

[0182]Each rabbit was placed in a restraint device and tagged with the
alphanumeric code. The formulations were instilled (50 μl) into the
conjunctival sac of both eyes 180, 120, 90 and 30 min before the first
paracentesis; then 15, 30, 90 min after the first paracentesis. To
perform the first paracentesis the animals were anaesthetized by
intravenous injection of 5 mg/kg Zoletil® (Virbac; 2.5 mg/kg
tiletamine HCl and 2.5 mg/kg zolazepam HCl) and one drop of local
anesthetic (Novesina®, Novartis) was administered to the eye.
Anterior chamber paracentesis was performed with a 26 G needle attached
to a tuberculin syringe; the needle was introduced into the anterior
chamber through the cornea, taking care not to damage the tissues. Two
hours after the first paracentesis, the animals were sacrificed with 0.4
ml Tanax® (Intervet International B.V.) and the second paracentesis
was performed. About 100 μl of aqueous humor were removed at the
second paracentesis. Aqueous humor was immediately split in four aliquots
and stored at -80° C. until analysis. Then both eyes were
enucleated and the iris-ciliary body was carefully excised, placed in
polypropylene tubes, and stored at -80° C. until analysis.

2.4.2 Pupillary Diameter Measurement

[0183]The pupillary diameter of both eyes was measured with a Castroviejo
caliper 180 min and 5 min before the first paracentesis and 5 min before
the second paracentesis.

2.4.3 Clinical Evaluation

[0184]The clinical evaluation of both eyes was performed by a slit lamp
(4179-T; Sbisa, Italy) at 180 min and 5 min before the first paracentesis
and 5 min before the second paracentesis. The clinical score was assigned
according to the following scheme:

[0185]0=normal

[0186]1=discrete dilatation of iris and conjunctival vessels

[0187]2=moderate dilatation of iris and conjunctival vessels

[0188]3=intense iridal hyperemia with flare in the anterior chamber

[0189]4=intense iridal hyperemia with flare in the anterior chamber and
presence of fibrinous exudates.

2.4.4 Prostaglandin E2 (PGE2) Measurement

[0190]For the quantitative determination of PGE2 in the aqueous humor
we used the PGE2 Immunoassay kit (R&D Systems; Cat. No. KGE004; Lot.
No. 240010). Eleven microliters or 16 μl of aqueous humor were diluted
to 110 μl or 160 μl with the calibrator diluent solution provided
with the kit. One hundred microliters of samples and of standards were
load into a 96-well plate and recorded in a plate layout. Samples were
treated following the assay procedure described in the kit. A microplate
reader (GDV, Italy; model DV 990 B/V6) set at 450 nm (wavelength
correction at 540 nm) was used for making the calibration and analyzing
the samples.

2.4.5 Protein Measurement

[0191]For protein concentration determination in the aqueous humor we used
the Protein Quantification Kit (Fluka; Cat. No. 77371; Lot. No. 1303129).
Five microliters of aqueous humor were diluted to 100 μl with water.
Twenty microliters of samples and of standards were load into a 96-well
plate and recorded in a plate layout. Samples were treated following the
assay procedure described in the kit. A microplate reader (GDV, Italy;
model DV 990 B/V6) set at 670 nm was used for making the calibration and
analyzing the samples.

2.4.6 Leukocytes (PMN) Measurement

[0192]For the determination of the number of leukocytes we used a
haemocytometer (Improved Neubauer Chamber; Bright-line, Hausser
Scientific) and a Polyvar 2 microscope (Reichert-Jung).

2.4.7 Leukotriene B4 (LTB4) Measurement

[0193]For the quantitative determination of LTB4 concentration in the
aqueous humor we used the LTB4 Immunoassay kit (R&D Systems; Cat.
No. KGE006; Lot. No. 243623). 11 μl of aqueous humor were diluted to
110 μl with the calibrator diluent solution provided with the kit. 100
μl of samples and of standards were load into a 96-well plate and
recorded in a plate layout. Samples were treated following the assay
procedure described in the kit. A microplate reader (GDV, Italy; model DV
990 B/V6) set at 450 nm (wavelength correction at 540 nm) was used for
making the calibration and analyzing the samples.

2.4.8 Myeloperoxidase (MPO) Measurement

[0194]The activity of MPO was measured as previously described by Williams
et al.[5] The iris-ciliary bodies were carefully dried, weighed and
immersed in 1 ml of hexa-decyl-trimethyl-ammonium bromide solution. Then,
the samples were sonicated for 10 sec on ice by a ultrasound homogenizer
(HD 2070, Bandelin electronic), freeze-thawed three times, sonicated for
10 sec and centrifuged at 14,000 g for 10 min to remove cellular debris.
An aliquot of the supernatant (40-2000 was diluted to 3 ml with the
o-dianisidine 2HCl/H2O2 solution. The change in absorbance at
460 nm was continuously monitored for 5 min by a spectrophotometer
(UV/Vis Spectrometer Lambda EZ 201; Perkin Elmer). The slope of the line
(Δ/min) was determined for each sample and used to calculate the
number of units of MPO in the tissue as follows:

MPOunit / g = ( Δ / min ) 10 6 μ
l mg ##EQU00001##

were ε=11.3 mM-1.Values were expressed as units of MPO/g of
tissue.

2.5 Data Analysis

[0195]Pupillary diameter, PGE2, protein, PMN, and MPO were expressed
as mean±SEM. Statistical analysis was performed using one way ANOVA
followed by a Newman-Keuls post hoc test. Clinical score was expressed as
% of eyes and the statistical analysis was performed using Kruskal-Wallis
followed by a Dunn post hoc test. P<0.05 was considered statistically
significant in both cases. Prism 4 software (GraphPad Software, Inc.) was
used for the analysis and graphs.

3. Results

3.1 Pupillary Diameter Measurement

[0196]The raw data are displayed in Tables T-2 and T-3. No statistical
significance was found between the CRT and all the treatments.

3.2 Clinical Evaluation

[0197]The raw data are displayed in Tables T-4 and T-5. Only the 0.5% LE
group showed a significant difference versus CTR (p<0.05).

[0199]The raw data are displayed in Tables T-8 and T-9. It has been found
a statistical significance for the treatments 0.03% F and 1% BOL vs CTR
with p<0.001, and 0.5% BOL vs CTR with p<0.05.

3.5 Leukocytes (PMN) Measurement

[0200]The raw data are displayed in Tables T-10 and T-11. All the
treatments were statistically significant vs CTR (p<0.001).

3.6 Leukotriene B4 (LTB4) Measurement

[0201]All samples were under the limit of quantification (about 0.2 ng/ml)
of the assay.

3.7 Myeloperoxidase (MPO) Measurement

[0202]The raw data are displayed in Tables T-12 and T-13. It has been
found a statistical significance for the all the treatments vs CTR with
p<0.01 for 0.03% F, and p<0.001 for 0.1% Dex, 0.5% LE, 0.1% BOL,
0.5% BOL and 1% BOL.

4. Discussion

[0203]The preliminary conclusions from the data generated are:
[0204]BOL-303242-X is active in this model. [0205]There was not a large
difference between these concentrations of BOL-303242-X and NSAID and
steroid positive controls.

[0206]There was not a profound dose-response for BOL-303242-X, perhaps
because we are at either maximal efficacy or maximal drug exposure at
these doses. However, the results show that BOL-303242-X is as effective
an anti-inflammatory drug as some of the commonly accepted prior-art
steroids or NSAID. Some other very preliminary data (not shown) suggest
that BOL-303242-X does not have some of the side effects of
corticosteroids.

[0212]Levels of cytokines associated with immune cells are direct
indications of activity of these cells in an inflammatory condition.
Reduced levels of these cytokines indicate a positive therapeutic effect
on inflammation of a test compound. This study was designed to determine
the effect of BOL-303242-X on IL-1β-induced cytokine production in
human corneal epithelial cells ("HCECs").

1. Purpose

[0213]To determine the effects of BOL-303242-X on IL-1β-stimulated
cytokine expression in primary human corneal epithelial cells using a
30-cytokine Luminex kit. Dexamethasone was used as a control.

3. Experimental Design

[0214]Primary HCECs were seeded in 24-well plates. After 24 h, cells were
treated with vehicle, IL-1β, IL-1β+dexamethasone, or
IL-1β+BOL-303242-X in basic EpiLife medium for 18 h (Table T-14).
Each treatment was performed in triplicate. Media were collected and used
for determination of cytokine content using a 30-cytokine Luminex kit.
Cell viability was determined by alamarBlue assay (LP06013).

[0215]Median fluorescence intensity (WI) was used to obtain the
concentration of each cytokines in pg/ml based on the standard curve of
each cytokine assayed by Luminex. The linear range of the standard curve
for each cytokine was used for determination of cytokine concentration.
Duplicate values for each sample were averaged. Data were expressed as
mean±SD. Statistical analysis was performed using one-way
ANOVA-Dunnett's test, and P<0.05 was considered statistically
significant.

5. Results

[0216]No statistically significant effect on cellular metabolic activity
(as measured by alamarBlue assay) was observed with the various
treatments.

[0217]Substantial amounts of 16 out of 30 cytokines tested were detected
in this study and 13 out of 14 cytokines detected were stimulated by 10
ng/ml IL-1β (Table T-14). IL-1β was excluded from analysis
because it was the stimulus. IL-1ra was excluded because the MFI was not
within the standard range.

[0218]Dexamethasone and BOL-303242-X significantly inhibited
IL-1β-stimulated cytokine production with comparable potency on 6
cytokines (IL-6, IL-7, MCP-1, TGF-α, TNF-α and VEGF), and a
significant inhibitory effect was observed at 1 nM on IL-6 and at 10 nM
on MCP-1, TGF-α and TNF-α (Table T-14 and FIGS. 1A-1F).

[0219]BOL-303242-X also significantly inhibited IL-1β-stimulated
G-CSF production with better potency compared to dexamethasone, and a
significant inhibitory effect was observed at 10 μg/mlby BOL-303242-X
while no significant effect was observed by dexamethasone on this
cytokine (FIG. 2).

[0220]BOL-303242-X also significantly inhibited IL-1β-stimulated
cytokine production with less potency compared to dexamethasone on 3
cytokines (GM-CSF, IL-8, and RANTES). A significant inhibitory effect was
observed at 1 nM by dexamethasone and at 10 nM by BOL-303242-X on GM-CSF.
A significant inhibitory effect was observed at 1 μM by dexamethasone
on RANTES while no significant effect was observed by BOL-303242-X on
this cytokine (FIGS. 3A-3C).

6. CONCLUSION

[0221]BOL-303242-X and dexamethasone have comparable potency for
inhibition of IL-1β-stimulated cytokine production in HCECs for the
cases of IL-6, IL-7, TGF-α, TNF-α, VGEF, and MCP-1.
BOL-303242-X is more potent than dexamethasone in inhibiting
IL-1β-stimulated production of G-CSF in HCECs. BOL-303242-X is
somewhat less potent than dexamethasone in inhibiting
IL-1β-stimulated production of GM-CSF, IL-8, and RANTES in HCECs.

Testing 3: Myocilin Expression in and Release from Trabecular Meshwork
Cells Upon Treatment with Dexamethasone or BOL-303242-X

Materials and Methods

TM Cells and Culture Media

[0222]All animal procedures were in accordance with the ARVO (Association
for Research in Vision and Ophthalmology) resolution on animal care. Eyes
from freshly killed, healthy rhesus monkeys (Macaca mulatta), obtained
from Lonza (Walkersville, Md.), were transported in CO2-independent
medium on ice, and processed approximately 40 hours post-enucleation.
Following removal of iris, lens, and the bulk of the ciliary body,
opercula (an anatomical feature of monkey TM) were stripped from anterior
segment quadrants. Using fine scissors, strips of TM were excised, and
subdivided TM fragments were explanted to multiwell plates containing
growth medium (described below) and incubated with Cytodex-3
gelatin-coated beads (Sigma Chemical Company, St. Louis, Mo.). The beads
attach to the explants within hours and provide additional substrate area
for out-migration of cells. Proliferating TM cells colonize additional
beads and also "spill" onto the tissue culture plastic and form colonies.
After several days, the original TM explants and beads were transferred
to new wells, generating additional primary cultures. Subconfluent
monolayers of cells on tissue culture plastic were passed from 12-well
plates to 35- or 60-mm dishes using a Collagenase-Dispase (Roche Applied
Bioscience, Indianapolis, Ind.). Second- or third-passage subcultures
were finally harvested enzymatically as above, and the cells were counted
and cryopreserved in liquid nitrogen.

[0224]For each study, up to nine TM cell strains, each derived from an
individual monkey, were tested separately. Cells were thawed and seeded
into 12- or 48-well clusters (Falcon, BD Biosciences; 150,000 and 30,000
cells/well, respectively) in proliferation medium. When cells were 75% to
90% confluent proliferation medium was replaced by a 5:4 mixture of HESFM
and Dulbecco's MEM, respectively, supplemented with 10% FBS, with added
taurine, ascorbic acid phosphate, glutathione, and antibiotic as for
proliferation medium (above), and with 2.72 g/L glucose and 1.72 g/L
fructose. At confluence, the medium was changed to Dulbecco's MEM,
containing 10% FBS40, ascorbic acid phosphate, antibiotic, 2.72 g/L
glucose and 1.72 g/L fructose. Cells were maintained as stable, confluent
monolayers in this latter medium for 4 to 7 days before experimental
treatments commenced.

TM Cell Treatments with DEX and BOL-303242-X

[0225]TM cell strains from nine different individual monkeys were used to
directly compare the responses to DEX and BOL-303242-X. Cells in
triplicate sample wells (24-well clusters) were incubated with DEX
(Sigma) in individual studies alongside corresponding cell samples
exposed to BOL-303242-X; drug concentrations ranged from 3 to 300 nM.

[0226]All treatments, including media for vehicle control samples,
contained a final DMSO concentration of 0.1% (v/v) across the
concentration ranges selected. Treatments lasted 96 hours, with one
exchange of medium on the third treatment day. The final 48-hr
conditioned media ("CM") samples were collected in their entirety (0.5
ml), centrifuged briefly to remove particulates, aliquoted, and stored at
-20° C. until thawed for analysis.

Cell Metabolic Activity Assay

[0227]A modification of previously described methods40 was employed
to evaluate cell metabolic activity, an index of cell viability. After
collection of CM samples, cells were briefly rinsed in modified Hanks
balanced salt solution containing Ca++ and Mg++ ("MHBSS"), and
then 0.0025% (w/v) resazurin (Sigma) in MHBSS was added to sample wells.
Plates were incubated (37° C., 5% CO2, 95% humidity) for 90
minutes, after which fluorescence (Excitation 560 nm, Emission 590 nm)
was read (Victor 3V Multilabel Counter, Wallac, Turku, Finland). As a
positive control for decreased cellular metabolic reduction of resazurin,
in each plate an additional well of vehicle control-treated cells was
preincubated with 0.06% hydrogen peroxide (Fisher, Atlanta, Ga.) in
MHBSS.

[0229]Following triplicate treatments with DEX, PA, BOL-303242-X, or
vehicle control medium, cultured TM cells prepared in 6-well clusters
were lysed, and total RNA was isolated using the RNeasy Plus MiniKit from
Qiagen (Valencia, Calif.) according to the manufacturer's instructions.
After quantification of purified total RNA (Quant-iT RNA Assay kit,
Molecular Probes, Eugene, Oreg.), equivalent amounts of this RNA were
apportioned to generate first-strand cDNAs for each treatment sample,
using random primers, (Affinity Script, Stratagene, La Jolla, Calif.).
Oligonucleotide myocilin primers, designed based on the cynomolgus MYOC
gene, and fluorescent Taqman probe (Applied Biosystems, Foster City,
Calif.) were used for PCR amplification. Equal amounts of total
RNA-equivalent mass (approximate range 250-1000 μg) reactant cDNA were
added to the PCR Master Mix (Stratagene) and myocilin primers/Taqman
probe. Amplification was performed in a thermocycler (Mx3005P,
Stratagene), with an initial denaturation step at 95° C. for 10
min, followed by 40 cycles of 95° C. for 15 sec and 60° C.
for 1 min for extension. Every run included standard controls (i.e.,
either without reverse transcriptase or lacking template). Relative
quantities of myocilin mRNA abundance were determined using differences
in threshold cycles ("Ct") between vehicle control and drug treatments.
Each sample was analyzed in triplicate wells, and the corresponding
values averaged for further quantitative analysis. Myocilin mRNA
abundance, expressed in proportion to vehicle control-treated samples,
was calculated using the Mx3005P software.

Data Analysis and Statistical Methods

[0230]Data underwent Box-Cox transformations for one- or two-way analysis
of variance (ANOVA), followed by the Tukey-Kramer test, using JMP
software (SAS, Cary, N.C.). The specific transformations used for
analyses are mentioned in the figure legends. For each set of triplicate
samples from the individual monkey TM cell strains tested, Western blot
densitometry values for myocilin protein detected in CM (as geometric
means), and relative abundance of myocilin mRNA (as geometric means),
were plotted as a function of drug concentrations. P-values less than
0.05 were considered statistically significant. Dose-response curve data
were fitted to a re-parameterized four-parameter logistic equation using
similar methodology to that previously described, and these equations
permitted estimation of the EC50 values±95% confidence intervals,
for each drug treatment.

Results

In Vitro Properties of Monkey TM Cells

[0231]Rhesus monkey TM cells demonstrated robust proliferation both in
primary explants and during early passage. General cellular morphology
and the uniform cobblestone pattern of the monolayers, consistent with TM
cells propagated from young human donors and cynomolgus monkeys reported
in the prior art, were maintained in confluent subcultures used for these
studies.

[0232]Effects of DEX and BOL-303242-X on Myocilin Protein in Monkey TM
Cell CM

[0233]Myocilin protein was released to CM by rhesus monkey TM cells, and
was detected in Western blots as a single thick band--probably a fused
doublet--at the expected molecular size, approximately 55 kDa, as
previously noted in Western blots of CM from DEX-treated human TM cells
and of monkey aqueous fluid. With exposure to increasing concentrations
of BOL-303242-X or DEX, immunoreactive bands of higher density could be
discerned by visual inspection alone. It is important to note though,
that DEX induced higher expression of myocilin than the BOL-303242-X at
high doses, suggesting a partial agonist activity for the BOL-303242-X on
myocilin gene expression.

[0234]FIG. 3 shows the effects of DEX and BOL-303242-X on the amount of
accumulated myocilin protein released into the CM during the second
48-hour treatment period. Whereas both compounds increased myocilin
concentrations in a dose-dependent manner, the amounts of myocilin
produced, and released into the medium, by BOL-303242-X, at all doses
studied, are less for BOL-303242-X than for DEX. As illustrated in FIG. 4
for one monkey TM cell strain, the full range of DEX treatments gave
statistically significant effects compared to vehicle control (FIG. 4,
solid symbols). (Note that 100 nM, corresponding to the topical dose
routinely used for DEX in clinical applications, is also commonly invoked
to assess steroid responsiveness in vitro55.) Within the dose range
utilized, maximal efficacy of DEX was achieved at 300 nM; for one of the
monkey TM cell strains that was tested this concentration of DEX yielded
a myocilin protein level 1233% (ca. 11-fold) over control. In several
strains tested, no clear plateau in the high concentration range was
identified for the DEX dose-response curve (FIG. 4). While BOL-303242-X
also increased myocilin accumulation in the CM of the monkey TM cells
throughout the concentration range tested (FIG. 4, open symbols), the
maximal efficacy computed across all nine TM cells strains, was about 50%
of that observed after DEX treatment (Table T-15). In fact, the dose
response curve for the BOL-303242-X showed clear indication of a plateau
approaching the high dose concentration range, indicating that the
compound had reached its maximal efficacy. The partial agonism of
BOL-303242-X was further demonstrated by the statistically significant
differences observed between DEX and BOL-303242-X at 3, 10, 100, and 300
nM (indicated by daggers in FIG. 4). With respect to potency, DEX and
BOL-303242-X displayed EC50s of 14.58 and 20.96 nM, respectively
(Table T-16). These differences were not statistically significant, with
overlapping 95% confidence limits for the estimates (Table T-16). In
experiments conducted over three months, the responses of the 9 monkey TM
isolates were similar and very reproducible; the inter-isolate
variabilities for the EC50s were 18.20% and 20.40% for DEX and
BOL-303242-X, respectively (Table T-16). The results indicate that
BOL-303242-X, as a partial GC agonist, induced significantly lower levels
of myocilin protein to be released by cultured monkey TM cells, compared
to the model GC DEX.

TABLE-US-00028
TABLE T-15
Partial agonism of BOL-303242-X in comparison with DEX.
Estimated efficacy at 300 nM, for inducing
myocilin protein expression in cultured monkey TM cells.
Efficacy ± SE1 Coef.
(weighted average; 95% Confidence of Variation
Compound %) Limits for Efficacy (%)
DEX 100 ± 6.09 88.07-111.93 18.27
BOL-303242-X 53.12 ± 2.20 48.81-57.43 12.42
1The efficacies presented are calculated as the weighted averages for
each experiment normalized to DEX (100%). The inverse of the variance for
each strain is used for the weight.
2Data are averaged from nine experiments (one per strain) that were
conducted over a period of three months.

TABLE-US-00029
TABLE T-16
Comparison of the potency of DEX and BOL-303242-X on
expression of myocilin protein by cultured monkey TM cells.
Compilation of data from two independent dose-response studies,
using nine monkey TM cell strains.
Coefficient of
95% Confidence Variation
Compound EC50 ± SE (nM)* Limits for EC50 (%)
DEX 14.58 ± 2.65 10.21-20.83 18.20
BOL-303242-X 20.96 ± 4.28 14.05-31.26 20.40
*The EC50s presented were calculated as the weighted averages of the
logarithm for the estimated EC50 for each TM cell strain in the
study. The inverse of the variance for the estimates was used for the
weight. The logarithms of the standard errors (SE) for the estimates were
converted back to the original scale using the Taylor series expansion.

Effects of DEX and BOL-303242-X on Myocilin mRNA Expression

[0235]The effects of DEX and BOL-303242-X on myocilin mRNA expression in
monkey TM are exemplified by the results shown in FIG. 5; data are from
the same cell strain depicted in FIG. 4 (above). The patterns for
expression of mRNA for myocilin were quite similar to those for protein,
in terms of the dose-response to DEX vs. BOL-303242-X (FIG. 5 panel),
also showing similar statistical significances to those observed for the
protein levels. The BOL-303242-X qRT-PCR data again were indicative of
the partial agonist nature of this agent, with significantly lower mRNA
abundance values at all doses compared with DEX. Maximal efficacy,
demonstrated at 300 nM for BOL-303242-X, was approximately 67% of that
for DEX (FIG. 5). Regarding estimated EC50s for all three drugs,
there was excellent general correlation between the values both for
myocilin protein and for mRNA abundance (Cf. Tables T-16 and T-17).
Indeed, as previously shown with myocilin protein in Table T-15, the
average (for n=4 strains) relative values for myocilin message were
significantly lower for BOL-303242-X vs. DEX at both 100 and 300 nM (FIG.
5; solid and open symbols for DEX- and BOL-303242-X-treated cells,
respectively).

TABLE-US-00030
TABLE T-17
Comparison of the potency of DEX and BOL-303242-X on
expression of myocilin mRNA in cultured monkey TM cells.
Compilation of data from two independent dose-response studies,
each using two monkey TM cell strains.
Coefficient of
95% Confidence Variation
Compound EC50 ± SE (nM)* Limits for EC50 (%)
DEX 14.66 ± 1.27 12.37-17.38 8.68
BOL-303242-X 20.75 ± 2.74 16.02-26.88 13.21
*The EC50s presented were calculated as the weighted averages of the
logarithm for the estimated EC50 for each TM cell strain in the
study. The inverse of the variance for the estimates was used for the
weight. The logarithms of the standard errors (SE) for the estimates were
converted back to the original scale using the Taylor series expansion.

Effects of Drugs on Cultured Monkey TM Cells in the Resazurin Reduction
Assay

[0236]There was no correlation of myocilin expression levels with general
cell metabolic status, as a consequence of exposure to different
concentrations of DEX or BOL-303242-X, nor did any drug treatments result
in a loss of cell viability compared to vehicle controls, as determined
by measuring chemical reduction of resazurin at the conclusion of the
treatment periods (results not shown). The results suggest, then, that
any increases or decreases observed in myocilin expression relative to
control, induced by any of the drug treatment regimens, were not due to
compromise of functional cell integrity.

[0237]Taken together, our results presented herein indicate that
BOL-303242-X exhibits a full agonist profile as an anti-inflammatory
agent and can have a more favorable therapeutic index than conventional
GCs when used for the treatment of ocular diseases with an inflammatory
component.

[0238]While specific embodiments of the present invention have been
described in the foregoing, it will be appreciated by those skilled in
the art that many equivalents, modifications, substitutions, and
variations may be made thereto without departing from the spirit and
scope of the invention as defined in the appended claims.

Patent applications by Bruce A. Pfeffer, Fairport, NY US

Patent applications by Charu A. Dewitt, Pittsford, NY US

Patent applications by Francisco J. Lopez, Victor, NY US

Patent applications by Keith W. Ward, Ontario, NY US

Patent applications by Mercedes Salvador-Silva, Rochester, NY US

Patent applications in class Nitrogen, other than as nitro or nitroso, attached directly to the six membered hetero ring by nonionic bonding

Patent applications in all subclasses Nitrogen, other than as nitro or nitroso, attached directly to the six membered hetero ring by nonionic bonding